Triacylglycerol synthesis in the liver is required to handle the influx of dietary and adipose-derived fatty acids, to convert dietary carbohydrate into storage lipids, and to package fatty acids into secreted very-low-density lipoprotein particles. When dietary fat and carbohydrate is excessive, hepatocytes store excess triacylglycerol, the liver becomes resistant to the action of insulin, and progression to steatohepatitis and liver damage may result. Gaps in our understanding of these processes linger because the major enzymes in the pathway of TAG synthesis have only recently been discovered and little is known about their individual functions and regulation. We are now in a position to bridge this gap with our recent identification of two new isoforms of glycerol-3-phosphate acyltransferase (GPAT), the committed step in the synthetic pathway. Why are three different GPAT isoforms needed in liver? We hypothesize that each isoform initiates the same pathway of glycerolipid synthesis, but that functional independence is necessary to separately regulate phospholipid and TAG synthesis, to regulate TAG synthesis under different physiological conditions, and to initiate nutrient-mediated signaling cascades involving mTOR and protein kinase C. We propose to investigate the functions of the three major GPAT isoforms in liver, GPAT1, 2 and 4. Aims 1 and 2 will focus on the functions of GPAT1, 2, and 4 in knockout mice and in cells, as well as the effects of GPAT gain-of-function and loss-of-function on TAG metabolism, lipoprotein production, hepatic steatosis, and insulin resistance. Aim 3 builds on intriguing new data that suggest that the pathway of TAG synthesis produces lipid intermediates with important signaling properties. We will use cultured cells to identify the signaling molecules that link tissue TAG content with insulin resistance. Narrative: The liver makes and stores fat normally, but when stored fat is present in excess amounts, the liver can become inflamed or have an impaired response to insulin. This application is designed to investigate the enzymes that initiate fat synthesis in the liver in order to understand how they are regulated and how they contribute to liver pathology.